SNARE proteins form the minimal machinery required for regulated exocytosis, a process that underlies all information transfer within the nervous system. Lipid rafts, which have been implicated in a number of diseases including Alzheimer's and Parkinson's, are dynamic membrane microdomains that serve as platforms to regulate membrane function. While SNARE proteins have been found to localize to lipid rafts, and while disruption of lipid rafts results in the inhibition of regulated exocytosis, the exact role that lipid rafts exert in mediating exocytosis has not yet been determined. This proposal seeks to: 1) identify the specific stages of regulated exocytosis (e.g., core complex formation, priming, and fusion) in which lipid rafts play an important role; 2) determine the function of lipid rafts in modulating fusion pore dynamics; and 3) determine the regulatory elements involved in targeting of the neuronal SNARE protein Syntaxin 1A to lipid raft domains. The experimental approach will utilize a combination of carbon fiber amperometry and membrane capacitance measurements in both intact and cholesterol-depleted chromaffin cells, as well as biochemical lipid raft isolations in HEK cells heterologously expressing various wild-type and mutant SNARE proteins.